The breakdown of fats, sugars, and other foods as well as
the synthesis of body constituents depends upon numerous
processes of making and breaking chemical bonds.
The cutting and forming of C—C bonds is especially challenging.
Enzymes can utilize chemical groupings of an
acidic or basic character that are present in the amino acids
from which the proteins are made. The acidic −COOH,
imidazolium (from histidine), and −NH3+ groups serve as
proton donors, and the unprotonated forms of these same
groups, as proton acceptors. These groups facilitate cleavage and formation of O—H, N—H, and C—H bonds. Certain
C—C bonds, e.g., those that are one atom removed from
a carbonyl group, can also be broken by proteins using
only their own catalytic acid–base groups. This is illustrated
in Fig. 13. The carbonyl group provides an electronaccepting
center into which electrons can flow temporarily
as the C—C bond is broken. Both the cleavage of a β
oxo alcohol and a β oxoacid (decarboxylation) are illustrated.
In some instances the carboxyl (—COOH) group
of a substrate can serve as an electron acceptor. However,
the reactivity toward bond cleavage is much higher
in a thioester such as that formed from acetyl-coenzyme A
(Fig. 10). This high reactivity accounts for one function of
coenzyme A. For example, coenzyme A permits the cleavage,
by a reverse Claisen condensation, of the fatty acid
chain during the β oxidation of fatty acids (Fig. 12, step e). However, not all C—C bonds can be broken using only
the chemical groupings of the proteins or of coenzyme A.

Participation of thiamin diphosphate or pyridoxal phosphate
is required for many other C—C bond cleavages.
Thiamin diphosphate enables cleavage of an α oxoacid as
indicated in Fig. 13. A characteristic of thiamin diphosphate
is that, when bound correctly into an active site, it
can lose a proton from its 5-membered thiazolium ring to
form the dipolar ionic “ylid” structure shown in Fig. 13.
This can add to the carbonyl group of an α oxoacid or
an α oxoalcohol to form a covalent compound (adduct)
in which the double bond of the thiazolium ring provides
the necessary electron-accepting center. The positive
change on the nitrogen atom of the ring assists in initiating
the chain cleavage. These thiamin-dependent cleavage
reactions are found in virtually every major metabolic
pathway in higher organisms and in most bacteria. For
example, the acetyl-CoA that is generated by β oxidation
of fatty acids (Fig. 12) enters the citric acid cycle where
the two carbon atoms of the acetyl group are converted
to CO2. One essential step in the cycle requires thiamin
diphosphate. It is hard to imagine how such metabolic
cycles could be organized without thiamin diphosphate.

Pyridoxal phosphate, sometimes in collaboration with
pyridoxamine phosphate, participates in dozens of different
reactions of amino acids, the building blocks of proteins.
These reactions involve both the biological synthesis
of amino acids and the breakdown of amino acids, e.g.,
of excess amino acids in the human diet. For these reactions,
the PLP is held in place by the enzyme in a location
adjoining the binding site for the specific amino acid substrate.
In this site an amino group of a protein side chain
(a lysine side chain; Protein −NH2) forms a Schiff base
linkage in which the carbonyl (C=O) group of PLP is converted
to a Schiff base linkage (C=N—Protein) similar to
that present in the PLP Schiff base drawn in Fig. 14. This
is the “resting form” of the enzyme. Then, in a two-step
process, the amino group of the substrate adds to the C=N
bond and displaces (eliminates) the Protein −NH2 group
to form the substrate Schiff base that is shown in generalized
form in Fig. 14. In this Schiff base, one of the
three bonds (a, b, c,) may be broken. This is illustrated
for cleavage a, removal of a hydrogen atom as H+ by a
catalytic group of the protein. The small arrows beside the
structure indicate the manner in which the pyridine ring of
the coenzyme, with a proton bound to the nitrogen atom
of its ring, serves as an electron acceptor in much the same
way as does thiamin diphosphate (Fig. 13). The structure
resulting from removal of the α-proton of the PLP Schiff
base is variously known as a “quinonoid” or “carbanionic”
intermediate. Depending upon the specificity of the enzyme
in whose site it is formed, this intermediate may
react in several ways. In a bacterial racemase a proton
may be returned to the α-carbon atom from which it was
removed but without stereospecificity, i.e., into either of
two positions relative to the other groups surrounding the
α-carbon. Some racemases are used by bacteria to convert
the stereoisomer known as L-alanine into the less common
“unnatural” D-alanine. The latter is incorporated into
the bacterial cell wall and helps provide protection to the
bacteria against attack by hydrolytic enzymes.

A second mode of reaction of the quinonoid-carbanionic
intermediate is utilizedbyplants whichsynthesize an
enzyme that acts on the amino acid S-adenosylmethionine
to form a cyclic three-membered ring compound aminocyclopropane
carboxylic acid. This is a major plant hormone.
In a third type of reaction a proton is added back to the
coenzyme itself (see Fig. 14) to form what is called a
ketimine (not illustrated). This is a Schiff base of pyridoxamine
phosphate (PMP, Fig. 5) with an α-oxoacid and is an
essential intermediate compound in the important process
of transamination (Fig. 14). This process is utilized by all
living organisms both in the synthesis of amino acids and
in the breakdown of excesses of amino acids. The human
body forms several amino acids via transamination. As
shown in Fig. 15, this is a reversible sequence involving a
cyclic interconversion of PLP and PMP in reaction steps
of the type illustrated in Fig. 14.

Figure 13 Activation of C—C bond cleavage by adjacent carbonyl
group (top) and by formation of adduct with thiamin diphosphate
(bottom).

Figure 14 The action of pyridoxal phosphate in initiating catalysis of numerous reactions of α-amino acids. Completion
of the various reactions requires a large variety of different enzyme proteins.

Figure 15 The transamination
reaction by which amino groups
are transferred from one carbon
skeleton (in the form of an α
oxoacid) to another to form or to
degrade an amino acid.

Yet another reaction for the ketimine illustrated in
Fig. 14 is the elimination of a substituent (labeled Y in this
drawing) with formation of a double bond. The product
of this elimination sometimes decomposes, with loss of
nitrogen as ammonia (NH3), but in other cases a molecule
carrying a different group may replace Y. Protonation of
the new Schiff base that results yields a new amino acid.
Several amino acids are made by plants and microorganisms
using this reaction sequence. Returning to the top of
Fig. 14, notice that cleavage of bond b leads to formation
of CO2 and decarboxylation of the substrate amino
acid. In this way the amino acid dihydroxyphenylalanine
(dopa) is converted to the neurotransmitter dopamine. The
latter can then be hydroxylated and methylated to form the
hormone adrenaline. Histidine is converted by decarboxylation
to histamine, a problem compound in allergic reactions,
while in the brain, the major excitatory neurotransmitter
is decarboxylated to gamma-aminobutyrate (gaba).
This is the major inhibitory transmitter in the central nervous
system and the compound that keeps our brains calm
enough to function. Cleavage of bond c (Fig. 14), when
R=H and Y=OH (the amino acid is serine) releases the
single-carbon compound formaldehyde. This process also
requires tetrahydrofolate (Fig. 6). In a converse type of
reaction glycine or serine may be condensed with various
carbonyl compounds to initiate new biosynthetic pathways.
These are often coupled to decarboxylation, which
helps to drive the sequence in the biosynthetic direction.
One of these yields the red heme pigment of blood.

A third coenzyme that is involved in C—C bond
cleavage and formation is the vitamin B12 derivative
5´-deoxyadenosylcobalamin (Fig. 7). In this compound
the cobalt–carbon bond is easily cleaved to form a free
radical which, in turn, facilitates C—C bond cleavage in
the substrate. The details, which are still under study, have
beenomitted, but Fig. 16 shows a general reaction in which
group X is often attached via a C—C bond which is broken.
The net result is that a hydrogen atom trades places with
group X. These rearrangement reactions, which cannot
be catalyzed by proteins alone or by other coenzymes,
are quite numerous in various bacteria. However, only
one of them occurs in human cells. That is the conversion
of methylmalonyl-CoA to succinyl-CoA, the reverse
of the succinyl-CoA mutase reaction as drawn in the
lower section of Fig. 16. The reaction is essential to the
metabolism of propionyl-CoA as is indicated at the bottom
of Fig. 12. Propionyl-CoA is carboxylated at the site
marked by an arrow in Fig. 17 to form methylmalonyl-
CoA. This compound must be isomerized by the vitamin
B12-dependent mutase to form succinyl-CoA which can be
oxidized to CO2 in the body’s central metabolic pathways.
Lack of the mutase is fatal.

Figure 16 (Top) A family of rearrangement reactions that depend
upon free radical formation involving an enzyme-bound form
of the vitamin B12 coenzyme 5´
-deoxyadenosylcobalamin (Fig. 7).
The rearrangement of (R) methylmalonyl-CoA to succinyl-CoA
(the opposite of the reaction shown here) is one of the two essential
vitamin B12-dependent reactions in the human body, and
plays an important role in fatty acid oxidation, as is indicated in
Fig. 12.

Figure 17 The carboxyl carrier function of biotin. A molecule of
activated CO2 is carried as —COOH bonded to N−1´
of biotin,
which is covalently attached (as in Fig. 11) to an appropriate
protein. Below this structure the sites of four different metabolic
intermediates that receive activated CO2 from carboxybiotin are
marked by arrows. In each case, either the thioester linkage to
coenzyme A or another adjacent carbonyl group activates a hydrogen
atom which dissociates as H+, leaving a negatively charged
site which accepts the CO2 by direct transfer from carboxybiotin.
Carboxylation of propionyl-CoA in the human body is an
essential step in degradation of branched chain and odd chainlength
fatty acids (Fig. 12). The resulting methylmalonyl-CoA is
converted to succinyl-CoA, the reverse of the reaction shown in
Fig. 16.